An increasing amount of biological assays, such as immunoassays and gene sequencing, are being carried out on micro arrays, such as DNA micro arrays or protein micro arrays. Micro arrays are also emerging as popular analytical tools for genomics and proteomics research. A microarray is a collection of microscopic spots containing probes, typically biological molecules such as DNA or protein spots attached to a solid planar surface, such as glass, plastic or silicon chip in a specific pattern and is used for analyzing biological interactions. Multiple probes can be assembled on a single substrate by techniques well know to one skilled in the art. A probe could bind to an analyte or group or analytes by hybridization or affinity binding. Examples of uses of such an array include, but are not limited to, investigations to determine which genes are active in cancer, investigations to determine which gene differences make a patient have a bad reaction to a drug treatment, investigations for infectious disease, investigations to determine presence of genetic mutation in a patient.
The in situ synthesis of micro arrays using solid-state chemistry and photolithography by a method called light-directed spatially addressable parallel chemical synthesis allows many micron-sized spots, each containing a unique protein/peptide sequence, to be simultaneously synthesized on a glass surface. This method uses a photolabile protection group to mask the N-terminus of an amino acid, and the glass surface during the peptide synthesis. Each deprotection and coupling cycle of the peptide synthesis is controlled by a set of photo masks with defined configurations that allow for the selection deprotection of the N-terminal amino group of the growing peptide chain, followed by selective coupling of different amino acids onto different peptides.
While DNA arrays have been quicker to develop and have emerged as a very powerful tool in genomics, there still exist bottlenecks in terms of the throughput of array synthesis as serial processes that involve manual intervention are used even when they are synthesized using photolithographic techniques. Proteins/peptides are fundamentally different from nucleic acids and the synthesis of protein/peptide arrays is much more complex than DNA arrays. The major impediment of using photolithography to generate high-density peptide arrays arises from the relatively high technical complexity need for peptide array construction with 20 amino acid building blocks, 20 photolabile protecting group containing amino acid derivatives and 20 different masks needed for each monomer elongation cycle. Therefore, the development of protein/peptide arrays has been slower and is still in its infancy. Whereas in the case of DNA arrays, only 4 masks are needed for each coupling cycle. Furthermore, peptide synthesis in general is much less efficient than the oligonucleotide synthesis, making it extremely difficult to generate high-quality peptide/protein arrays.
In generally, depending on the method by which the microarray is created, it can be (a) in situ photolithographic array, (b) in situ SPOT synthesized array, and (c) contact printing (also called spotting) array.
The chemistry of the in situ photolithographic array uses light directed parallel chemical synthesis and solid-state chemistry. This approach is limited largely due to the inefficient photochemical reaction needed throughout the whole synthesis. As a result, only short peptides (or peptide analogs, e.g., peptoids) can be sufficiently synthesized by the in situ photolithographic synthesis approach.
The SPOT-synthesis approach is also by in situ synthesis, but it does not use photochemical reactions for deprotection of the N-terminal amino group of the growing peptide chain. The SPOT-synthesis comprises the dispensing of a small volume of solutions containing Fmoc-amino acids and other coupling reagents to a designated stop on a membrane. Subsequently, deprotection and coupling steps synthesize the biomolecule on the substrate to form protein/peptide array.
The contact printing array method makes use of an automatic spotter to spot nanoliter droplets of pre-synthesized peptide/protein solutions onto a suitably derivatized solid surface, e.g., glass surface. By this approach, each peptide/protein is synthesized only once in a bulk quantity, and multiple spots containing the peptide/protein are created by printing using a spotter.
The more preferred methods for making protein/peptide arrays are contact printing and SPOT-synthesis. The SPOT-synthesis and contact printing methods permit rapid and highly parallel synthesis of huge numbers of proteins/peptides and proteins/peptide mixtures (pools) including a large variety of unnatural building blocks, as well as a growing range of other organic compounds. Yet, the major drawbacks of these methods for synthesizing biomolecule micro arrays are their low throughput, low degree of automation, low density of target molecules, low yield and batch to batch and process variability. Also, these method are not as miniaturized as the in situ photolithography technique for microarray synthesis.
In short, the current methods of manufacturing micro array rely on serial processing/synthesis of micro arrays using highly customized tool sets such as peptide/DNA synthesizers, spotters, ink jet printers. They involve manual processing operations not amenable to low cost, automated, high throughput, high volume manufacturing.